The search for new therapeutic agents has been greatly aided in recent years by a better understanding of the structure of enzymes and other biomolecules associated with diseases. One important class of enzymes that has been the subject of extensive study is protein kinases.
Protein kinases constitute a large family of structurally related enzymes that are responsible for the control of a variety of signal transduction processes within the cell. (See, Hardie, G. and Hanks, S. The Protein Kinase Facts Book, I and II, Academic Press, San Diego, Calif.: 1995). Protein kinases are thought to have evolved from a common ancestral gene due to the conservation of their structure and catalytic function. Almost all kinases contain a similar 250–300 amino acid catalytic domain. The kinases may be categorized into families by the substrates they phosphorylate (e.g., protein-tyrosine, protein-serine/threonine, lipids, etc.). Sequence motifs have been identified that generally correspond to each of these kinase families (See, for example, Hanks, S. K., Hunter, T., FASEB J. 1995, 9, 576–596; Knighton et al., Science 1991, 253, 407–414; Hiles et al., Cell 1992, 70, 419–429; Kunz et al., Cell 1993, 73, 585–596; Garcia-Bustos et al., EMBO J. 1994, 13, 2352–2361).
In general, protein kinases mediate intracellular signaling by effecting a phosphoryl transfer from a nucleoside triphosphate to a protein acceptor that is involved in a signaling pathway. These phosphorylation events act as molecular on/off switches that can modulate or regulate the target protein biological function. These phosphorylation events are ultimately triggered in response to a variety of extracellular and other stimuli. Examples of such stimuli include environmental and chemical stress signals (e.g., osmotic shock, heat shock, ultraviolet radiation, bacterial endotoxin, and H2O2), cytokines (e.g., interleukin-1 (IL-1) and tumor necrosis factor α (TNF-α)), and growth factors (e.g., granulocyte macrophage-colony-stimulating factor (GM-CSF), and fibroblast growth factor (FGF)). An extracellular stimulus may affect one or more cellular responses related to cell growth, migration, differentiation, secretion of hormones, activation of transcription factors, muscle contraction, glucose metabolism, control of protein synthesis, and regulation of the cell cycle.
Many diseases are associated with abnormal cellular responses triggered by protein kinase-mediated events as described above. These diseases include, but are not limited to, cancer and other proliferative disorders. Accordingly, there has been a substantial effort in medicinal chemistry to find protein kinase inhibitors that are effective as therapeutic agents.
The c-Met proto-oncogene encodes the c-Met receptor tyrosine kinase. The c-Met receptor is a 190 kDa glycosylated dimeric complex composed of a 50 kDa alpha chain disulfide-linked to a 145 kDa beta chain. The alpha chain is found extracellularly while the beta chain contains extracellular transmembrane and cytosolic domains. c-Met is synthesized as a precursor and is proteolytically cleaved to yield mature alpha and beta subunits. It displays structural similarities to semaphorins and plexins, a ligand-receptor family that is involved in cell-cell interaction. The ligand for c-Met is hepatocyte growth factor (HGF), a member of the scatter factor family and has some homology to plasminogen [Longati, P. et al., Curr. Drug Targets 2001, 2, 41–55); Trusolino, L. and Comoglio, P. Nature Rev. Cancer 2002, 2, 289–300].
c-Met functions in tumorigenesis and tumor metastasis. Chromosomal rearrangements forming Tpr-met fusions in an osteoclast cell line resulted in constitutively active c-Met receptors and transformation (Cooper, C. S. et al., Nature 1984, 311, 29–33). c-Met mutants exhibiting enhanced kinase activity have been identified in both hereditary and sporadic forms of papillary renal carcinoma (Schmidt, L. et al., Nat. Genet. 1997, 16, 68–73; Jeffers, M. et al., Proc. Nat. Acad. Sci. 1997, 94, 11445–11500). Expression of c-Met along with its ligand HGF is transforming, tumorigenic, and metastatic (Jeffers, M. et al., Oncogene 1996, 13, 853–856; Michieli, P. et al., Oncogene 1999, 18, 5221–5231). HGF/Met has been shown to inhibit anoikis, suspension-induced programmed cell death (apoptosis), in head and neck squamous cell carcinoma cells. Anoikis resistance or anchorage-independent survival is a hallmark of oncogenic transformation of epithelial cells (Zeng, Q. et al., J. Biol. Chem. 2002, 277, 25203–25208).
c-Met is overexpressed in a significant percentage of human cancers and is amplified during the transition between primary tumors and metastasis. To investigate whether this oncogene is directly responsible for the acquisition of the metastatic phenotype, Giordano et al. exploited a single-hit oncogenic version of c-Met that was able to transform and to confer invasive and metastatic properties to nontumorigenic cells, both in vitro and in nude mice. They found a point mutation in the signal transducer docking site of c-Met that increased the transforming ability of the oncogene, but abolished its metastatic potential. They concluded that the metastatic potential of the c-Met oncogene relies on the properties of its multifunctional docking site, and that a single point mutation affecting signal transduction can dissociate neoplastic transformation from metastasis. Giordano, S., et al, Proc. Nat. Acad. Sci. 94: 13868–13872, 1997.
c-Met is implicated in various cancers. One cancer type in which c-Met is implicated is gastric adenocarcinoma. The American Cancer Society has projected that in 2004, 22,710 people will be diagnosed with gastric cancer and 11,780 will die of the disease in the United States. The five-year survival rate for patients who present with late stage disease involving the proximal region of the stomach is only 10–15%.
c-Met is amplified in 19–39% of gastric adeno-carcinomas, with highest amplification rates seen in diffuse type gastric adenocarcinoma. Tahara, E. (2004) Genetic Pathways of Two Tpes of Gastric Cancer. IARC Sci Publ. 2004, (157): 327–49. c-Met is also over-expressed in approximately 70% of gastric adenocarcinomas as examined by immunohistochemistry with over-expression correlating with tumor stage. Heideman D. A. et al., 2001, J Pathol 194: 428–435; Absence of tpr-met and expression of c-Met in human gastric mucosa and carcinoma; Amemiya, H. et al., 2002, c-Met Expression in Gastric Cancer with Liver Metastasis Oncology 63: 286–296. The potential for autocrine activation of c-Met exists in approximately 50% of patients as their tumors also express HGF3.
c-Met is also implicated in renal cancer. It was found that the beta-subunit of the c-Met protooncogene product is the cell-surface receptor for hepatocyte growth factor. It was also identified that the hepatocyte growth factor receptor is the c-Met protooncogene product. Bottaro, D. P., et al, 1991, Science 251: 802–804.
c-Met is also implicated in small cell lung carcinoma. Small cell lung cancer is an aggressive disease that has a 5-year survival of 5–10% (mostly limited disease patients). In 2004, the number of new cases of lung cancer, which includes both small cell and non-small cell types, is 173,770 with 160,440 patients dying from the disease.
In 2003, small cell lung cancer made up approximately 16% of all lung cancer cases. Jemal A., et al., 2003, Cancer statistics, 2003. CA Cancer J. Clin., 53: 5–26; Ma P. C., et al., 2003, c-MET mutational analysis in small cell lung cancer: novel juxtamembrane domain mutations regulating cytoskeletal functions. Cancer Res. 63: 6272–81. Both c-Met and c-Kit are co-expressed in the majority of small cell cancer cell lines and tumors and 30% of cell lines examined harbor mutations in c-Met. Rygaard K, et al., 1993, Expression of the proto-oncogenes c-Met and c-Kit and their ligands, hepatocyte growth factor/scatter factor and stem cell factor, in SCLC cell lines and xenografts. Br J Cancer. 67(1): 37–46. HGF is co-expressed in a small number of tumors.
The nexus between c-Met and colorectal cancer has also been established. Analysis of c-Met expression during colorectal cancer progression showed that 50% of the carcinoma specimens analyzed expressed 5–50-fold higher levels of c-Met mRNA transcripts and protein versus the adjacent normal colonic mucosa. In addition, when compared to the primary tumor, 70% of colorectal cancer liver metastasis showed c-Met over expression. See Long et al., 2003, Met Receptor Overexpression and Oncogenic Kiras Mutation Cooperate to Enhance Tumorigenicity of Colon Cancer Cells in Vivo. Mol Cancer Res. March; 1(5): 393–401; Fujisaki, et al., 1999, CD44 stimulation induces integrin-mediated adhesion of colon cancer cell lines to endothelial cells by up-regulation of integrins and c-Met and activation of integrins. Cancer Res. September 1;59 (17): 4427–34; Hiscox et al., Association of the HGF/SF receptor, c-Met, with the cell-surface adhesion molecule, E-cadherin, and catenins in human tumor cells. Biochem Biophys Res Commun. 1999, Aug. 2; 261(2): 406–11; Herynk et al., 2003, Activation of c-Met in colorectal carcinoma cells leads to constitutive association of tyrosine-phosphorylated beta-catenin. Clin Exp Metastasis. 20(4): 291–300; Wielenga et al., Expression of c-Met and heparan-sulfate proteoglycan forms of CD44 in colorectal cancer. Am J Pathol. 2000 November;157(5): 1563–73; Di Renzo et al., 1995, Overexpression and amplification of the Met/HGF receptor gene during the progression of colorectal cancer. Clin. Cancer Res., 1: 147–154; and Mao, et al., 1997, Activation of c-Src by receptor tyrosine kinases in human colon cancer cells with high metastatic potential. Oncogene, 15:3083–3090.
The c-Met receptor tyrosine kinase (“RTK”) is also implicated in glioblastoma. High-grade malignant gliomas are the most common cancers of the central nervous system., Despite treatment with surgical resection, radiation therapy, and chemotherapy, the mean overall survival is <1.5 years, and few patients survive for >3 years. A common reason for treatment failure is their innate resistance to radiation and chemotherapy.
Glioblastoma multiforme is the most common and most malignant glial neoplasm. Despite very aggressive treatment, these malignant gliomas are associated with an average life expectancy of only 9 months. The formation and malignant progression of human gliomas are complex processes and involve genetic mutations, chromosomal multiploidy, and aberrant epigenetic influences of multiple mitogens and angiogenic factors.
Human malignant gliomas frequently express both HGF and c-Met, which can establish an autocrine loop of biological significance. Glioma c-Met expression correlates with glioma grade, and an analysis of human tumor specimens showed that malignant gliomas have a 7-fold higher HGF content than low-grade gliomas.
On top of representing the most common form of primary central nervous system malignancy, gliomas are also among the tumors most tightly linked with HGF-cMet signaling abnormalities. Multiple studies have demonstrated that human gliomas frequently co-express HGF and c-Met and that high levels of expression are associated with malignant progression. HGF gene transfer to glioma cell lines enhances tumorigenicity, tumor growth, and tumor-associated angiogenesis. It has also been shown that blocking HGF-cMet signaling reverses these phenotypes in vivo. It was further shown that HGF-cMet is able to activate Akt and protect glioma cell lines from apoptotic death, both in vitro and in vivo. See Hirose et al., Clinical importance of c-Met protein expression in high-grade astrocytic tumors. Neurol. Med.-Chir. 38:851–859, 1998, Hirose et al., 1998, Immunohistochemical examination of cMet protein expression inastrocytic tumors. Acta Neuropathol. 95:345–351; Koochekpour et al., Met and hepatocyte growth factor expression in human gliomas. Cancer Res. 57:5391–5398; Laterra et al., HGF expression enhances human glioblastoma tumorigenicity and growth. Biochem. Biophys. Res. Commun. 235:743–747; Moriyama et al., 1995, Concomitant expression of hepatocyte growth factor, HGF activator and cMet genes in human glioma cells in vitro. FEBs Lett. 372:78–82; Nabeshima et al., Expression of cMet correlates with grade of malignancy in human astrocytic tumors: an immunohistochemical study. Histopathology 31:436–443, 1997, Shiota et al., Coexpression of hepatocyte growth factor and its receptor (cMet) in HGL4 glioblastoma cells. Lab. Investig. 53:511–516, 1996, Welch et al., Hepatocyte growth factor and receptor (cMet) in normal and malignant astrocytic cells. Anticancer Res. 19:1635–1640, 1999, Bowers et al., 2000, HGF protects against cytoxic death in human glioblastoma via PI3-K and Akt-dependent pathways. Cancer Res. 60:4277–4283.
It was shown that the effect of NK4 (HGF antagonist), on HGF-promoted growth of a human breast cancer resulted in the reduction of tumor invasiveness and motility, weight and volume. Furthermore, in the in-vitro invasion assay and migration assay, both HGF and human fibroblasts, which secrete bioactive HGF, increased the invasiveness and migration of the breast cancer cells (MDA MB 231). See Growth and angiogenesis of human breast cancer in a nude mouse tumor model is reduced by NK4, the HGF antagonist. Carcinogenesis, May 9, 2003. Furthermore, transgenic mice harboring mutationally activated c-Met developed metastic mammary carcinoma. These same activating mutants were able to establish tumors in nude mouse NIH 3T3 xenografts (PNAS, Vol 95, pp 14417–14422, November 1998).
Transgenic mice that overexpressed c-Met in hepatocytes developed heptocellular carcinoma (HCC), one of the human tumors in which c-Met has been implicated previously. Inactivation of the transgene led to regression of even highly advanced tumors, apparently mediated by apoptosis and cessation of cellular proliferation. Numerous cells were proliferating in the liver tumors that were elicited by c-Met. Removal of the stimulus from the transgenic hMet led to prompt cessation of cellular proliferation even in the cells of advanced malignancies (The Journal of Cell Biology, Vol. 153, 2001, p. 1023–1033).
HGF/Met signaling is involved in cell adhesion and motility in normal cells and plays a major role in the invasive growth that is found in most tissues, including cartilage, bone, blood vessels, and neurons (reviewed in Comoglio, P. M. and Trusolino, L. J. Clin. Invest. 2002, 109, 857–862). Dysfunctional activation or increased numbers of c-Met is likely to contribute to the aberrant cell-cell interactions that lead to migration, proliferation, and survival of cells that is characteristic of tumor metastasis. Activation of c-Met induces and sustains a variety of tumors [Wang, R. et al., J. Cell. Biol. 2001, 153, 1023–1034; Liang, T. J. et al., J. Clin. Invest. 1996, 97, 2872–2877; Jeffers, M. et al., Proc. Nat. Acad. Sci. 1998, 95, 14417–14422] while loss of c-Met inhibits growth and invasiveness of tumor cells [Jiang, W. G. et al., Clin. Cancer Res. 2001, 7, 2555–2562; Abounader, R. et al., FASEB J. 2002 16, 108–110]. Increased expression of Met/HGF is seen in many metastatic tumors including colon (Fazekas, K. et al., Clin. Exp. Metastasis 2000, 18, 639–649), breast (Elliott, B. E. et al., 2002, Can. J. Physiol. Pharmacol. 80, 91–102), prostate (Knudsen, B. S. et al., Urology 2002, 60, 1113–1117), lung (Siegfried, J. M. et al., Ann. Thorac. Surg. 1998, 66, 1915–1918), and gastric (Amemiya, H. et al., Oncology 2002, 63, 286–296).
Further demonstration of the role c-Met plays in metastasis was shown by Giordano, et al. (2002) who presented evidence for cross-talk between the semaphorin 4D (SEMA4D; 601866) receptor, plexin B1 (PLXNB1; 601053), and c-Met during invasive growth in epithelial cells. Binding of SEMA4D to PLXNB1 stimulated tyrosine kinase activity of MET, resulting in tyrosine phosphorylation of both receptors. This effect was not found in cells lacking c-Met expression. Giordano, S., et al: 2002, The Semaphorin 4D receptor controls invasive growth by coupling with Met. Nature Cell Biol. 4: 720–724.
HGF-c-Met signaling has also been associated with increased risk of atherosclerosis (Yamamoto, Y. et al., J. Hypertens. 2001, 19, 1975–1979; Morishita, R. et al., Endocr. J. 2002, 49, 273–284) and increased fibrosis of the lung (Crestani, B. et al., Lab. Invest. 2002, 82, 1015–1022).
The Janus kinases (JAK) are a family of tyrosine kinases consisting of JAK1, JAK2, JAK3 and TYK2. The JAKs play a critical role in cytokine signaling. The downstream substrates of the JAK family of kinases include the signal transducer and activator of transcription (STAT) proteins. JAK/STAT signaling has been implicated in the mediation of many abnormal immune responses such as allergies, asthma, autoimmune diseases such as transplant rejection, rheumatoid arthritis, amyotrophic lateral sclerosis and multiple sclerosis as well as in solid and hematologic malignancies such as leukemias and lymphomas. The pharmaceutical intervention in the JAK/STAT pathway has been reviewed [Frank Mol. Med. 5: 432–456 1999 & Seidel, et al, Oncogene 19: 2645–2656 2000].
JAK1, JAK2, and TYK2 are ubiquitously expressed, while JAK3 is predominantly expressed in hematopoietic cells. JAK3 binds exclusively to the common cytokine receptor gamma chain (gc) and is activated by IL-2, IL-4, IL-7, IL-9, and IL-15. The proliferation and survival of murine mast cells induced by IL-4 and IL? 9 have, in fact, been shown to be dependent on JAK3- and gc-signaling [Suzuki et al, 2000, Blood 96: 2172–2180].
Cross-linking of the high-affinity immunoglobulin (Ig) E receptors of sensitized mast cells leads to a release of proinflammatory mediators, including a number of vasoactive cytokines resulting in acute allergic, or immediate (type I) hypersensitivity reactions [Gordon et al, 1990; Nature 346: 274–276 & Galli, 1993, N. Engl. J. Med., 328: 257–265]. A crucial role for JAK3 in IgE receptor-mediated mast cell responses in vitro and in vivo has been established [Malaviya, et al, 1999, Biochem. Biophys. Res. Commun. 257: 807–813]. In addition, the prevention of type I hypersensitivity reactions, including anaphylaxis, mediated by mast cell-activation through inhibition of JAK3 has also been reported [Malaviya et al, 1999; J. Biol. Chem. 274:27028–27038]. Targeting mast cells with JAK3 inhibitors modulated mast cell degranulation in vitro and prevented IgE receptor/antigen-mediated anaphylactic reactions in vivo.
A recent study described the successful targeting of JAK3 for immune suppression and allograft acceptance. The study demonstrated a dose-dependent survival of Buffalo heart allograft in Wistar Furth recipients upon administration of inhibitors of JAK3 indicating the possibility of regulating unwanted immune responses in graft versus host disease [Kirken, transpl. proc. 33: 3268–3270 2001].
IL-4-mediated STAT-phosphorylation has been implicated as the mechanism involved in early and late stages of rheumatoid arthritis (RA). Up-regulation of proinflammatory cytokines in RA synovium and synovial fluid is a characteristic of the disease. It has been demonstrated that IL-4-mediated activation of IL-4/STAT pathway is mediated through the Janus Kinases (JAK 1 & 3) and that IL-4-associated JAK kinases are expressed in the RA synovium [Muller-Ladner, et al, 2000, J. Immunol. 164: 3894–3901].
Familial amyotrophic lateral sclerosis (FALS) is a fatal neurodegenerative disorder affecting about 10% of ALS patients. The survival rates of FALS mice were increased upon treatment with a JAK3 specific inhibitor. This confirmed that JAK3 plays a role in FALS [Trieu, et al, 2000, Biochem. Biophys. Res. Commun. 267: 22–25].
Signal transducer and activator of transcription (STAT) proteins are activated by, among others, the JAK family kinases. Results form a recent study suggested the possibility of intervention in the JAK/STAT signaling pathway by targeting JAK family kinases with specific inhibitors for the treatment of leukemia [Sudbeck, et al, 1999, Clin. Cancer Res. 5: 1569–1582]. JAK3 specific compounds were shown to inhibit the clonogenic growth of JAK3-expressing cell lines DAUDI, RAMOS, LC1; 19, NALM-6, MOLT-3 and HL-60.
In animal models, TEL/JAK2 fusion proteins have induced myeloproliferative disorders and in hematopoietic cell lines, introduction of TEL/JAK2 resulted in activation of STAT1, STAT3, STAT5, and cytokine-independent growth [Schwaller, et al, 1998, EMBO J. 17: 5321–5333].
Inhibition of JAK 3 and TYK 2 abrogated tyrosine phosphorylation of STAT3, and inhibited cell growth of mycosis fungoides, a form of cutaneous T cell lymphoma. These results implicated JAK family kinases in the constitutively activated JAK/STAT pathway that is present in mycosis fungoides [Nielsen, et al, Proc. Nat. Acad. Sci. U.S.A. 94: 6764–6769 (1997)]. Similarly, STAT3, STAT5, JAK1 and JAK2 were demonstrated to be constitutively activated in mouse T cell lymphoma characterized initially by LCK over-expression, thus further implicating the JAK/STAT pathway in abnormal cell growth [Yu, et al, 1997, J. Immunol. 159: 5206–5210]. In addition, IL-6-mediated STAT3 activation was blocked by an inhibitor of JAK, leading to sensitization of myeloma cells to apoptosis [Catlett-Falcone, et al, 1999; Immunity 10: 105–115].
Tyrosine kinases are a class of enzymes that mediate intracellular signal transduction pathways. Abnormal activity of these kinases has been shown to contribute to cell proliferation, carcinogenesis and cell differentiation. Thus, agents that modulate the activity of tyrosine kinases are useful for preventing and treating proliferative diseases associated with these enzymes.
KDR is a tyrosine kinase receptor that also binds VEGF (vascular endothelial growth factor) Neufeld et al., 1999, FASEB J., 13, 9. The binding of VEGF to the KDR receptor leads to angiogenesis, which is the sprouting of capillaries from preexisting blood vessels. High levels of VEGF are found in various cancers causing tumor angiogenesis and permitting the rapid growth of cancerous cells. Therefore, suppressing VEGF activity is a way to inhibit tumor growth, and it has been shown that this can be achieved by inhibiting KDR receptor tyrosine kinase. For example, SU5416 is a selective inhibitor of the tyrosine kinase and was reported to also suppress tumor vascularization and the growth of multiple tumors. Fong et al., 1999, Cancer Res. 59, 99. Other inhibitors of KDR tyrosine kinase for the treatment of cancer have also been reported (WO 98/54093, WO 99/16755, WO 00/12089).
Examples of cancers that may be treated by such inhibitors include brain cancer, genitourinary tract cancer, lymphatic system cancer, gastric cancer, cancer of the larynx, lung cancer, pancreatic cancer, breast cancer, Kaposi's sarcoma, and leukemia. Other diseases and conditions associated with abnormal tyrosine kinase activity include vascular disease, autoimmune diseases, ocular conditions, and inflammatory diseases.
As a result of the biological importance of protein kinases, there is current interest in therapeutically effective protein kinase inhibitors. Accordingly, there is still a great need to develop inhibitors of protein kinases that are useful in treating various diseases or conditions associated with protein kinase activation. In particular, it would be desirable to develop compounds that are useful as inhibitors of c-Met, JAK, and KDR particularly given the inadequate treatments currently available for the majority of the disorders implicated in their activation.